The subject of the present invention is a system for expressing a Toxoplasma gondii surface antigen, the antigen thus obtained and its use for diagnostic and/or therapeutic purposes.
Toxoplasmosis is an infectious disease caused by a Toxoplasma gondii protozoal parasite, a member of the class Sporozoa and of the order Coccidia. Toxoplasma gondii is an intracellular parasite which reproduces in a wide variety of cell types inside its hosts, which are mammals.
This parasite, which is very widespread geographically, is an important pathogen, not only in human medicine, but also in veterinary medicine.
In man, two forms of the parasite have been described: the xe2x80x9ctachyzoitexe2x80x9d, which is the multiplicative form encountered during the acute phase of the disease and the xe2x80x9cbradyzoitexe2x80x9d, a resistant form which persist encysted in the nervous tissues, and which is probably responsible for maintaining a durable immunity to reinfection.
In humans, toxoplasmosis is most often asymptomatic and most often passes unnoticed without any consequences. There are however cases in which a Toxoplasma infection or a reactivation of a previously acquired infection can generate serious disorders for the so-called at risk individuals who are pregnant women and immunodepressed or immunosuppressed subjects. This organism has multiple replication sites. However, it may be responsible for severe cerebral and ocular impairments when its replication site is the cells of the central nervous system and the cells of the reticuloendothelial system. Pregnant women represent high-risk subjects, since a Toxoplasma infection, especially during the first few months of pregnancy, may be responsible for serious foetal and neonatal complications if maternal treatment is not undertaken early and pursued assiduously. In particular, newborns contaminated via the transplacental route are subject to serious ocular and cerebral disorders which are even fatal in certain cases. Immunodepressed patients and particularly AIDS patients are subject to serious toxoplasmosis due most often to reactivations of previous infections.
It was therefore essential to have available diagnostic tests which make it possible to determine the presence of the parasite, especially in pregnant women, either by detecting antibodies which may be present in the individual, or by detecting the presence of Toxoplasma antigens in the subject.
HUGUES in xe2x80x9cCurrent topics in Microbiology and Immunologyxe2x80x9d, Vol. 120 (1985), SPRINGER Ed., pages 105-139 has listed a number of commercially available serodiagnostic tests such as the SABIN and FELDMAN staining test, standardized by BEVERLY and BEATTLE in 1958 and perfected by FELDMAN and LAMB (1966), WALDELAND (1976) and BALFOUR et al. (1982); the REMINGTON (1968) test for the detection of antibodies by immunofluorescence, optimized in 1975 by KARIM and LUDLAM; the hemagglutination tests; the ELISA test for the detection of antibodies specific for Toxoplasma, by the isolation of IgM in situ on a microplate described in 1983 by WIELARRD et al.
The different tests used are based on the detection of antibodies or antigens specific for toxoplasmosis. One of the critical points therefore consisted in the characterization of the Toxoplasma gondii major antigens which induce a specific immune response and are capable of being used in serological detection tests.
In this regard, Burg et al. (Abstract c85 J. Cell. Biochem., 1986, 1017, 145) have described the exploitation of an expression library in E. coli using the complementary DNA (cDNA) obtained from Toxoplasma gondii messenger RNAs and the isolation, from this bank, of sequences encoding the antigens present at the surface of the parasite, with the aid of polyclonal antisera directed against the purified surface antigenic proteins P30 and P22
Some authors have shown that P30 constitutes the major surface antigen (see Kasper et al., J. Immunol. 1983, 130, 2407-2412) and can be used for the production of vaccines or in diagnostic tests especially in immunoassays. Moreover, Boothroyd et al. (see Patent Application WO 89/08700) have identified and obtained the genetic material encoding Toxoplasma gondii P30 protein and suggest the use of the gene for the production of recombinant protein, peptides and antibodies. This gene has been cloned (Burg et al., 1988, J. Immunol., 141, 3584-3591). Analysis of the sequence shows a secretory signal positioned at the N-terminal end which is cleaved in the mature P30 protein and a highly hydrophobic C-terminal region which is also cleaved and replaced by a glycolipid which allows its membrane anchorage, and a potential N-glycosylation site.
There remains, however, a problem which consists in producing sufficient quantities of the P30 antigen both for the preparation of vaccines and for use in immunological tests. Kim et al. (Infection and Immunity, January 1994, 62, 203-209) have described the expression of a recombinant P30 with a conformation similar to that of the native protein in CHO cells without, however, succeeding in obtaining satisfactory expression levels.
There is, moreover, an advantage in producing a P30 which is secreted into the culture medium in order to facilitate its production and its use for the preparation of diagnostic tests or of pharmaceutical compositions.
Consequently, the subject of the present invention is an expression cassette which is functional in a cell derived from a nonmammalian eukaryotic organism allowing the expression of a DNA fragment encoding a Toxoplasma gondii P30 protein, placed under the control of the elements necessary for its expression, said P30 protein being secreted from said cell derived from a eukaryotic organism and recognized by human antisera.
In general, any cell derived from a nonmammalian eukaryotic organism can be used within the framework of the present invention and most particularly an insect cell or a cell derived from a lower eukaryotic organism. The term xe2x80x9ccell derived from a lower eukaryotic organismxe2x80x9d refers to a cell derived from a unicellular or pluricellular eukaryotic organism not possessing a mechanism which allows cell differentiation. Such cells are known to a person skilled in the art. There will be preferably used, however, a fungus, especially a unicellular fungus, or a yeast, especially of the strain Kluyveromyces, Pichia, Hasegawaea, Saccharomyces or Schizosaccharomyces and most particularly selected from the group consisting of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Schizosaccharomyces malidevorans, Schizosaccharomyces sloofiae, Schizosaccharomyces octosporus and Hasegawaea japonicus. Of course, these examples are not limiting. A large number of these cells are commercially available in collections such as ATCC (Rockville, Mass., USA) and AFRC (Agriculture and Food Research Council, Norfolk, UK).
For the purposes of the present invention, said cell may be of the wild type or mutant type. In this context, an auxotrophic mutant cell is particularly preferred which has lost the capacity to synthesize at least one metabolite essential for its growth, such that it can grow only in a medium supplemented with this specific metabolite or by complementation with a gene allowing its synthesis. Although a large number of auxotrophy mutations for various essential metabolites are currently known, there may be mentioned more particularly the mutations inhibiting the synthesis of arginine, of leucine and of uracil. Such mutations are described in the literature which is accessible to persons skilled in the art. A large number of auxotrophy mutations affecting various biosynthesis pathways can generally be generated by applying the following approach. Briefly, it is sufficient to cultivate culture the wild-type cells treated with the aid of a mutagen, at the same time in the presence and in the absence of the targeted essential metabolite and to search for the mutants which will multiply only in its presence contrary to the wild-type cells which can grow in both cases.
An expression cassette according to the invention is intended for the production of a P30 protein in a form which is secreted from the cell derived from a non-mammalian eukaryotic organism and which is recognized by anti-Toxoplasma antisera. Such antisera are obtained from patients who have contracted a recent or distant toxoplasmosis containing immunoglobulins which recognize the Toxoplasma gondii antigens and especially P30. It goes without saying that said P30 protein can also be recognized by other antibodies directed against the natural P30 protein, such as for example monoclonal or polyclonal antibodies obtained by immunizing a variety of species with the natural protein.
P30 protein is understood to mean the Toxoplasma gondii surface antigen produced by the genetic recombination techniques described in the present application or any fragment or mutant of this antigen provided that it is immunologically reactive with antibodies directed against the P30 protein of this parasite. Advantageously, such a protein has an amino acid sequence exhibiting a degree of homology of at least 70%, preferably of at least 85% and, most preferably, of at least 95% compared with the sequence specified by Burg et al. (1988, supra). In practice, such an equivalent may be obtained by deletion, substitution and/or addition of one or more amino acids of the native protein. Persons skilled in the art know the techniques which make it possible to carry out these modifications without affecting immunological recognition.
A DNA fragment in use for the purposes of the present invention may be obtained by any technique in use in the field of the art, for example by cloning a Toxoplasma gondii genomic DNA library or a complementary DNA (cDNA) library with an appropriate probe, by PCR (Polymerase Chain Reaction) or alternatively by chemical synthesis.
Within the framework of the present invention, use will be made of a P30 protein in which all or part of the hydrophobic C-terminal region has been amputated and which comprises the elements appropriate to allow secretion, such as for example a secretory signal. Of course, the latter may be homologous, that is to say derived from the native P30 protein. In this context, a preferred expression cassette according to the invention will allow the production of a P30 protein having the sequence as shown in the sequence identifier NO: 1 starting with the amino acid +1 and ending with the amino acid +299. As mentioned above, it may also be a mutant or a fragment of said P30 protein.
Alternatively, a heterologous secretory signal, that is to say one derived from any secreted or membrane protein, can also be used provided, however, that it is functional in the considered cell derived from a non-mammalian eukaryotic organism. According to this variant, an expression cassette according to the invention comprises a DNA fragment encoding a P30 protein having the sequence as shown in the sequence identifier NO: 1 starting with the amino acid +31 and ending with the amino acid +299 or a fragment or mutant of said P30 protein, said DNA fragment comprising, in addition, a sequence encoding a heterologous secretory signal. The latter is generally placed upstream of the first N-terminal residue of the mature P30 protein, namely the residue 31 of SEQ ID NO: 1. The choice of a secretory signal is wide and is accessible to persons skilled in the art. By way of example, there may be mentioned that of the major acid phosphatase (pho1) of Schizosaccharomyces pombe (Elliot et al., 1986, J. Biol. Chem. 261, 2936-2941) and the pre-pro sequence of the alpha sex pheromone (Mating Factor alpha or MFxcex1) of Saccharomyces cerevisiae (Kurjan and Herskowitz, 1982, Cell, 30, 933-934).
According to an advantageous embodiment, an expression cassette according to the invention allows the production of a nonglycosylated Toxoplasma gondii P30 protein. Although any conventional method can be used to inhibit N-glycosylation in the considered cell derived from a nonmammalian eukaryotic organism, such as for example the addition of tunicamycin to the culture medium, it is preferred, however, to mutate the potential N-glycosylation site so that it is no longer recognized by the cellular enzymes participating in the glycosylation. To this end, there is preferably used a DNA fragment encoding a P30 protein comprising at least one mutation, said mutation being characterized by the presence of an amino acid residue different from the natural residue in position 241 and/or 243 of the sequence as shown in the sequence identifier NO: 1, provided that the amino acid residue in position 243 is not a threonine. A mutant of the P30 protein which is particularly preferred within the framework of the present invention comprises a glutamine residue in position 241, in place of the natural asparagine residue.
Of course, an expression cassette according to the invention may allow the production of a P30 protein (having an amino acid sequence as specified above) fused with an exogenous component which can aid its stability, its purification or its production. The choice of such a component is wide and accessible to persons skilled in the art. It may especially be an exogenous protein or peptide. There may be mentioned for example the pho1 protein of Schizosaccharomyces pombe, xcex2-galactosidase, a chain of lysine residues (poly Lys) or histidine residues (poly His). The fusion may take place at the N- or C-terminus of the P30 protein in use in the present invention.
An expression cassette according to the invention comprises elements necessary for the expression of said DNA fragment in the considered cell derived from a nonmammalian eukaryotic organism. xe2x80x9cComponents necessary for the expressionxe2x80x9d is understood to mean all the elements which allow the transcription of a DNA fragment into messenger RNA (mRNA) and the translation of the latter into protein. Among these, the promoter region is of special importance. It can be constitutive, that is to say it can allow a constant transcription level during the entire cell cycle. By way of nonlimiting examples, the promoter regions derived from the genes PGK (3-phosphoglycerate kinase) and MFxcex1 of Saccharomyces cerevisiae (Hitzeman et al., 1983, Science, 219, 620-625), adh (alcohol dehydrogenase) of Schizosaccharomyces pombe (Russel and Hall, 1983, J. Biol. Chem., 258, 143-149) and the baculovirus p39K early promoters (Guarino et al., 1986, J. Virol., 57, 563) and pl2.5K late promoters (Hill-Perkins et al., 1990, J. Gen. Virol., 71, 971-976).
However, it may be advantageous to use a regulable promoter region which makes it possible to vary the transcription levels according to the culture conditions or the cell growth phase depending on the presence of an inducer (activation of transcription) or of a repressor (repression). In general, the regulable promoter regions are derived from regulable genes for which the regulation mechanisms may be highly varied. As regards Schizosaccharomyces pombe, there may be mentioned the thermal shock genes whose expression increases with temperature (Gallo et al., 1991, Mol. Cell. Biol., 11, 281-288) and the gene encoding the enzyme fructose diphosphatase (fdp) whose transcription is repressed in the presence of glucose and induced under conditions of deficiency (Hoffman and Winston, 1989, Gene, 84, 473-479). Also entering into this category are the genes which are regulable by thiamine such as the genes pho4 (Yang and Schweingruber, 1990, Curr. Genet., 18, 269-272), nmt1 (Maundrell, 1990, J. Biol. Chem., 265, 10857-10864) and thi2 (Zurlinden and Schweingruber, 1992, Gene, 117, 141-143) whose expression is regulated at the transcriptional level by thiamine, more precisely repressed in the presence of thiamine and induced or derepressed in its absence.
As regards a regulable promoter region, a promoter region regulable by thiamine is preferably most particularly used. The latter may be isolated, by conventional techniques, from genes corresponding to this type of regulation, such as those mentioned above. Of course, it can be modified by mutation, deletion and/or addition of one or more nucleotide(s) compared with the sequence of the native promoter region, provided, however, that these modifications do not drastically alter its regulatory capacity. A fragment of such a promoter region can also be used, especially a fragment comprising the activation and/or repression sequences responsible for the regulation by thiamine. This fragment is placed upstream of a conventional TATA box and a conventional site of initiation of transcription which are capable of initiating transcription in the considered cell derived from a nonmammalian eukaryotic organism. Although a single fragment of the promoter region is sufficient to ensure the regulation by thiamine, it is also possible to envisage, in order to enhance the levels of expression, using several fragments placed in tandem and in any orientation relative to the TATA box.
One particularly advantageous expression cassette according to the invention is that which combines a promoter region derived from the Schizosaccharomyces pombe pho4 gene and a DNA fragment encoding a P30 protein as defined above.
On the other hand, an expression cassette according to the invention may, in addition, contain other elements which contribute to the expression of the DNA fragment, especially a transcription termination sequence such as that of the Schizosaccharomyces pombe arg3 gene (Van Huffel et al., 1992, Eur. J. Biochem., 205, 33-43) as well as transcription activation sequences.
According to a particularly advantageous embodiment, an expression cassette according to the invention allows the production, in a form secreted from the nonmammalian cell, of at least 0.1 mg/l of a P30 protein, advantageously of at least 0.2 mg/l and preferably of at least 0.3 mg/l.
The present invention also extends to a vector comprising an expression cassette according to the invention. This may be a viral vector and especially a vector derived from a baculovirus, most particularly intended for expression in an insect cell.
It may also be an autonomously replicating plasmid vector and in particular a multicopy vector present between 5 and 500 copies in the host cell, advantageously between 10 and 400 copies and, preferably, between 20 and 300 copies. There may be mentioned, by way of example, the vectors derived from pGEM3 (Weilguny et al., 1991, Gene, 99, 47-54) and pFL20 (Losson and Lacroute, 1983, Cell, 32, 371-377). Moreover, a vector according to the invention may also comprise elements which ensure its replication, such as the origin 2 of Saccharomyces cerevisiae or ars of Schizosaccharonyces pombe and, optionally, ori of Escherichia coli. In addition, it may also comprise a selectable gene, such as (i) a gene allowing the synthesis of an essential metabolite, especially the URA3 or LEU2 gene of Saccharomyces cerevisiae and the ura4 or leu1 gene of Schizosaccharomyces pombe or (ii) an antibiotic resistance gene.
The present invention also relates to a cell derived from a nonmammalian eukaryotic organism comprising an expression cassette according to the invention either in a form integrated into the cell genome or inserted into a vector. A cell according to the invention has been defined above. There are preferred most particularly an insect cell, a unicellular fungus or a yeast, especially a yeast selected from the group consisting of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Schizosaccharomyces malidevorans, Schizosaccharomyces sloofiae, Schizosaccharomyces octosporus and Hasegawaea japonicus. 
The present invention also relates to a P30 protein produced by an expression cassette, a vector or a cell derived from a nonmanmalian eukaryotic organism according to the invention. Within the framework of the present invention, the P30 protein may be modified in vitro, especially by the addition or deletion of chemical groups, such as phosphates, sugars or myristic acid so as to enhance its stability or the presentation of one or more epitope(s)
The subject of the present invention is also a process for the preparation of a P30 protein, according to which:
(i) a cell derived from a nonmammalian eukaryotic organism is cultured under appropriate conditions; and
(ii) said protein secreted from said cell derived from a nonmammalian eukaryotic organism is recovered.
Within the framework of the invention, the P30 protein is recovered directly in the culture medium according to conventional purification techniques such as for example hydrophobic interaction or ion-exchange chromatography, gel filtration or immunopurification.
As regards the variant according to which the P30 protein is produced using an expression cassette comprising a promoter region regulable by thiamine and, in particular, derived from the Schizosaccharomyces pombe pho4 gene, the cells harboring such a cassette are cultured in a medium supplemented with thiamine when the culture is intended solely for their propagation. As soon as a culture is carried out in order to produce a Toxoplasma gondii P30 protein, the cells are transferred to a thiamine-free medium. Such a variant is more particularly intended to be carried out with lower eukaryotic cells. Thus, according to a process of the invention:
(i) a cell derived from a lower eukaryotic organism is cultured under appropriate conditions in the absence of thiamine, and
(ii) said protein secreted from said cell derived from a lower eukaryotic organism is recovered.
The present invention also relates to a reagent for the detection and/or the monitoring of a Toxoplasma gondii infection, which comprises, as reactive substance, a recombinant protein as defined above.
The above reagent is or will be attached directly or indirectly onto an appropriate solid support. The solid support may be, without limitation, in the form of a cone, a tube, a well, beads or the like.
The term xe2x80x9csolid supportxe2x80x9d as used here includes all materials on which a reagent may be immobilized for use in diagnostic tests. Natural or synthetic materials, chemically modified or otherwise, can be used as solid support, especially polysaccharides such as cellulose materials, for example paper, cellulose derivatives such as cellulose acetate and nitrocellulose; polymers such as vinyl chloride, polyethylene, polystyrenes, polyacrylate or copolymers such as propylene and vinyl chloride polymer, vinyl chloride and vinyl acetate polymer; styrene-based copolymers, natural fibers such as cotton and synthetic fibers such as nylon.
Preferably, the solid support is a polystyrene polymer or a butadiene-styrene copolymer. Advantageously, the support is a polystyrene or a styrene-based copolymer comprising between about 10 and 90% by weight of styrene units.
The attachment of the reagent onto the solid support may be performed in a direct or indirect manner.
In the direct manner, two approaches are possible: either by adsorption of the reagent onto the solid support, that is to say by non-covalent bonds (mainly of the hydrogen, Van der Waals or ionic type), or by establishing covalent bonds between the reagent and the support.
In the indirect manner, an xe2x80x9canti-reagentxe2x80x9d compound capable of interacting with the reagent can be attached beforehand (by adsorption or covalent bonding) onto the solid support in order to immobilize the whole onto the solid support. By way of example, there may be mentioned an anti-P30 antibody, provided that it is immunologically reactive with a part of the protein which is different from that involved in the reaction for recognition of the antibodies of the sera; a ligand receptor system, for example by grafting onto the P30 protein a molecule such as a vitamin and by immobilizing, on the solid phase, the corresponding receptor (for example the biotin-streptavidin system). Indirect manner is also understood to mean the preliminary grafting or the insertion, by genetic recombination, of a protein or of a polypeptide at one end of the P30 protein and the immobilization of the latter on the solid support by passive adsorption or covalent bonding of the protein or of the polypeptide.
The invention also relates to a process for the detection of anti-Toxoplasma antibodies in a biological sample, such as a blood sample, from an individual or from an animal likely to be or to have been infected by Toxoplasma gondii, according to which at least the following steps are performed:
a mixture is prepared comprising:
i) a reagent as defined above which is or which will be immobilized on a solid support,
ii) the sample,
iii) a labeled anti-immunoglobulin;
the mixture is incubated for a predetermined time;
the solid phase is separated from the liquid phase; and
the possible presence of anti-Toxoplasma antibodies is revealed by measuring the level of labeling in the solid phase.
In one embodiment of the invention,
a mixture is prepared comprising:
i) the reagent immobilized on the solid support, and
ii) the sample;
the mixture is incubated for a predetermined time which allows the formation of an immune complex immobilized on the solid support;
an anti-immunoglobulin labeled under appropriate incubating conditions allowing its reaction with the immobilized immune complex is added;
the solid phase is separated from the liquid phase; and
the possible presence of anti-Toxoplasma antibodies is revealed by measuring the level of labeling in the solid phase.
In another embodiment of the invention, the process for the detection of anti-Toxoplasma antibodies in a biological sample, such as a blood sample, from an individual or an animal likely to be or to have been infected by Toxoplasma gondii comprises at least the following steps:
a mixture is prepared comprising:
i) an anti-immunoglobulin which is or which will be attached onto a solid support;
ii) the sample;
iii) a labeled reagent which comprises, as reactive substance, the abovementioned P30 protein;
the mixture is incubated for a predetermined time;
the liquid phase is separated from the solid phase; and
the possible presence of anti-Toxoplasma antibodies is revealed by measuring the level of labeling in the solid phase.
Advantageously, a mixture is prepared, comprising the anti-immunoglobulin attached onto the solid support and the sample,
the mixture is incubated for a predetermined time allowing the formation of an immune complex immobilized onto the solid support;
the liquid phase is separated from the solid phase;
the labeled reagent is added which comprises, as reactive substance, the abovementioned P30 protein; and
the possible presence of anti-Toxoplasma antibodies is revealed by measuring the level of labeling in the solid phase.
By way of example, there may be mentioned, as marker, an enzyme such as horseradish peroxidase, alkaline phosphatase, a radioactive isotope such as 125I, 3H, 57Co, a luminescent marker and the like.
The invention also relates to monoclonal or polyclonal antibodies obtained by the immunological reaction of a human or animal organism to an immunogenic agent consisting of the recombinant P30 protein and their use, as reactive substance, in a reagent for the detection and/or monitoring of a Toxoplasma gondii infection in a biological sample, such as a tissue sample; the antibodies being previously labeled with any appropriate marker as defined above.
Accordingly, the subject of the invention is a process for the detection of the Toxoplasma gondii P30 protein in a biological sample, such as a tissue sample, from an individual or an animal likely to be or to have been infected by Toxoplasma gondii, according to which the sample and a reagent as defined above are brought into contact under appropriate conditions which allow a possible immunological reaction, and the possible presence of an immune complex formed with the above mentioned reagent is detected by measuring the level of labeling in the biological sample.
The subject of the invention is also an active immunotherapeutic composition, especially a vaccinal preparation, which comprises, as active ingredient, a recombinant P30 protein, the active ingredient being optionally conjugated with a pharmaceutically acceptable support, and optionally an excipient and/or an appropriate adjuvant.
The present invention also covers a pharmaceutical composition intended for the treatment or for the prevention of a Toxoplasma gondii infection in man or animals, comprising a therapeutically effective quantity of an expression cassette, a vector, a cell derived from a nonmammalian eukaryotic organism or a P30 protein according to the invention or prepared according to a process according to the invention.